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Modified two-component gelation systems, methods of use and methods of manufacture

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US8741326B2
US8741326B2 US11561328 US56132806A US8741326B2 US 8741326 B2 US8741326 B2 US 8741326B2 US 11561328 US11561328 US 11561328 US 56132806 A US56132806 A US 56132806A US 8741326 B2 US8741326 B2 US 8741326B2
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alginate
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Gene Michal
Shubhayu Basu
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Abbott Cardiovascular Systems Inc
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    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K33/00Medicinal preparations containing inorganic active ingredients
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    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18Growth factors; Growth regulators
    • A61K38/1858Platelet-derived growth factor [PDGF]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36Blood coagulation or fibrinolysis factors
    • A61K38/363Fibrinogen
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    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46Hydrolases (3)
    • A61K38/48Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482Serine endopeptidases (3.4.21)
    • A61K38/4833Thrombin (3.4.21.5)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION, OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14Macromolecular materials
    • A61L27/26Mixtures of macromolecular compounds
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION, OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3873Muscle tissue, e.g. sphincter
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION, OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS, OR SURGICAL ARTICLES
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52Hydrogels or hydrocolloids
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    • C12YENZYMES
    • C12Y304/00Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21Serine endopeptidases (3.4.21)
    • C12Y304/21005Thrombin (3.4.21.5)

Abstract

A bioscaffolding can be formed from a mixture of gel components of different gelation systems. For example, a bioscaffolding can be formed by mixing at least two different components of at least two different two-component gelation systems to form a first mixture and by mixing at least two different components (other than the components that make up the first mixture) of the at least two different two-component gelation systems to form a second mixture. A treatment agent, such as a cell type or a growth factor, can be added to either the first mixture or the second mixture. In some embodiments, the treatment agent is not added to either mixture. The first mixture can be co-injected with the second mixture to form a bioscaffolding in an infarct region for treatment thereof.

Description

FIELD OF INVENTION

Post-myocardial infarction treatments and compositions.

SEQUENCE LISTING

An electronic copy of the Sequence Listing entitled “5618P5128_SeqList_ST25” is incorporated herein by reference. This Sequence Listing consists of [SEQ ID NOs: 1-3].

BACKGROUND OF INVENTION

Ischemic heart disease typically results from an imbalance between the myocardial blood flow and the metabolic demand of the myocardium. Progressive atherosclerosis with increasing occlusion of coronary arteries leads to a reduction in coronary blood flow. “Atherosclerosis” is a type of arteriosclerosis in which cells including smooth muscle cells and macrophages, fatty substances, cholesterol, cellular waste product, calcium and fibrin build up in the inner lining of a body vessel. “Arteriosclerosis” refers to the thickening and hardening of arteries. Blood flow can be further decreased by additional events such as changes in circulation that lead to hypoperfusion, vasospasm or thrombosis.

Myocardial infarction (MI) is one form of heart disease that often results from the sudden lack of supply of oxygen and other nutrients. The lack of blood supply is a result of a closure of the coronary artery (or any other artery feeding the heart) which nourishes a particular part of the heart muscle. The cause of this event is generally attributed to arteriosclerosis in coronary vessels.

Formerly, it was believed that an MI was caused from a slow progression of closure from, for example, 95% then to 100%. However, an MI can also be a result of minor blockages where, for example, there is a rupture of the cholesterol plaque resulting in blood clotting within the artery. Thus, the flow of blood is blocked and downstream cellular damage occurs. This damage can cause irregular rhythms that can be fatal, even though the remaining muscle is strong enough to pump a sufficient amount of blood. As a result of this insult to the heart tissue, scar tissue tends to naturally form.

Various procedures, including mechanical and therapeutic agent application procedures, are known for reopening blocked arteries. An example of a mechanical procedure includes balloon angioplasty with stenting, while an example of a therapeutic agent application includes the administration of a thrombolytic agent, such as urokinase. Such procedures do not, however, treat actual tissue damage to the heart. Other systemic drugs, such as ACE-inhibitors and Beta-blockers, may be effective in reducing cardiac load post-MI, although a significant portion of the population that experiences a major MI ultimately develop heart failure.

An important component in the progression to heart failure is remodeling of the heart due to mismatched mechanical forces between the infarcted region and the healthy tissue resulting in uneven stress and strain distribution in the left ventricle. Once an MI occurs, remodeling of the heart begins. The principle components of the remodeling event include myocyte death, edema and inflammation, followed by fibroblast infiltration and collagen deposition, and finally scar formation from extra-cellular matrix (ECM) deposition. The principle component of the scar is collagen which is non-contractile and causes strain on the heart with each beat. Non-contractility causes poor pump performance as seen by low ejection fraction (EF) and akinetic or diskinetic local wall motion. Low EF leads to high residual blood volume in the ventricle, causes additional wall stress and leads to eventual infarct expansion via scar stretching and thinning and border-zone cell apoptosis. In addition, the remote-zone thickens as a result of higher stress which impairs systolic pumping while the infarct region experiences significant thinning because mature myocytes of an adult are not regenerated. Myocyte loss is a major etiologic factor of wall thinning and chamber dilation that may ultimately lead to progression of cardiac myopathy. In other areas, remote regions experience hypertrophy (thickening) resulting in an overall enlargement of the left ventricle. This is the end result of the remodeling cascade. These changes also correlate with physiological changes that result in increase in blood pressure and worsening systolic and diastolic performance.

SUMMARY OF INVENTION

A bioscaffolding can be formed from a mixture of gel components of different gelation systems. In some embodiments, a bioscaffolding can be formed by mixing at least two different components (which do not gel upon mixing) of at least two different two-component gelation systems to form a first mixture and by mixing at least two different components (other than the components that make up the first mixture and which do not gel upon mixing) of the at least two different two-component gelation systems to form a second mixture. A treatment agent, such as a cell type or a growth factor, can be added to either the first mixture or the second mixture. The first mixture can then be co-injected with the second mixture to form a bioscaffolding in an infarct region for treatment thereof. The first and second mixtures can be co-injected with a dual-lumen delivery device, which can include, but are not limited to, a dual syringe, a dual-needle left-ventricle injection device, a dual-needle transvascular wall injection device and the like.

In some embodiments, a bioscaffolding can be formed by mixing at least two different gelation components (which do not gel upon mixing) to form a first mixture. A treatment agent, such as a cell type or a growth factor, can be added to the first mixture. The first mixture can then be co-injected with a second gelation component to form a bioscaffolding on or within an infarct region for treatment thereof. The first mixture and the gelation component can be co-injected with, a dual-lumen delivery device, which can include, but are not limited to, a dual syringes a dual-needle left-ventricle injection device, a dual-needle transvascular wall injection device and the like.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1B illustrate the progression of heart damage once the build-up of plaque in an artery induces an infarct to occur.

FIG. 2 illustrates an embodiment of a dual bore delivery device.

FIGS. 3A-3B illustrate an alternative embodiment of a dual bore delivery device.

FIGS. 4A-4C illustrate a second alternative embodiment of a dual bore delivery device.

DETAILED DESCRIPTION

FIGS. 1A-1B illustrate the progression of heart damage once the build-up of plaque induces an infarct to occur. FIG. 1A illustrates a site 10 where blockage and restricted blood flow can occur from, for example, a thrombus or embolus. FIG. 1B illustrates resultant damage area 20 to the left ventricle that can result from the lack of oxygen and nutrient flow carried by the blood to the inferior region left of the heart. The damage area 20 will likely undergo remodeling, and eventually scarring, resulting in a non-functional area.

Bioscaffoldings formed of two components and applied in situ to the left heart ventricle can be used to treat post-myocardial infarction tissue damage. “Bioscaffolding” and “two-component gelation system” and “gelation system” are hereinafter used interchangeably. Examples of two-component gelation systems include, but are not limited to, alginate construct systems, fibrin glues and fibrin glue-like systems, self-assembled peptides and combinations thereof. Each component of the two-component gelation system may be co-injected to an infarct region by a dual-lumen delivery device. Examples of dual-lumen delivery devices include, but are not limited to, a dual syringe, dual-needle left-ventricle injection devices, dual-needle transvascular wall injection devices and the like.

In some embodiments, at least one cell type may be co-injected with at least one component of the two-component gelation system to an infarct region. In some embodiments, the cells may be mixed with at least one component of the two-component gelation system before the two-components are co-injected to the infarct region. Examples of cell types, include, but are not limited to, localized cardiac progenitor cells, mesenchymal stem cells, bone marrow derived mononuclear cells, adipose stem cells, embryonic stem cells, umbilical cord blood derived stem cells, smooth muscle cells or skeletal myoblasts.

In some applications, the two-component gelation system includes a fibrin glue. Fibrin glue consists of two main components, fibrinogen and thrombin. Fibrinogen is a plasma glycoprotein of about 340 kiloDaltons (kDa) in its endogenous state. Fibrinogen is a symmetrical dimer comprised of six paired polypeptide chains, alpha, beta and gamma chains. On the alpha and beta chains, there is a small peptide sequence called a fibrinopeptide which prevent fibrinogen from spontaneously forming polymers with itself. In some embodiments, fibrinogen is modified with proteins. Thrombin is a coagulation protein. When combined in equal volumes, thrombin converts the fibrinogen to fibrin by enzymatic action at a rate determined by the concentration of thrombin. The result is a biocompatible gel which gelates when combined at the infarct region. Fibrin glue can undergo gelation between about 5 to about 60 seconds. Examples of other fibrin glue-like systems include, but are not limited to, Tisseel™ (Baxter), Beriplast P™ (Aventis Behring), Biocol® (LFB, France), Crosseal™ (Omrix Biopharmaceuticals, Ltd.), Hemaseel HMN® (Haemacure Corp.), Bolheal (Kaketsuken Pharma, Japan) and CoStasis® (Angiotech Pharmaceuticals).

In some applications, the two-component gelation system includes self-assembled peptides. Self-assembled peptides generally include repeat sequences of alternating hydrophobic and hydrophilic amino acid chains. The hydrophilic amino acids are generally charge-bearing and can be anionic, cationic or both. Examples of cationic amino acids are lysine and arginine. Examples of anionic amino acids are aspartic acid and glutamic acid. Examples of hydrophobic amino acids are alanine, valine, leucine, isoleucine or phenylalanine. Self-assembled peptides can range from 8 to 40 amino acids in length and can assemble into nanoscale fibers under conditions of physiological pH and osmolarity. In sufficient concentration and over time, the fibers can assemble into an interconnected structure that appears macroscopically as a gel. Self-assembled peptides typically undergo gelation between several minutes to several hours. Examples of self-assembled peptides include, but are not limited to: AcN-RARADADARARADADA-CNH2 (SEQ ID NO: 1) (RAD 16-II) wherein R is arginine, A is alanine, D is aspartic acid, and Ac indicates acetylation; VKVKVKVKV-PP-TKVKVKVKV-NH2 (SEQ ID NO: 2) (MAX-1) wherein V is valine, K is lysine and P is proline; and AcN-AEAEAKAKAEAEAKAK-CNH2 (SEQ ID NO: 3) wherein A is alanine, K is lysine and E is glutamic acid (EAK 16-II). Self-assembled peptides show good cytocompatibility, as represented by cell adhesion, cell migration and proliferation.

In some applications, the two-component gelation system is an alginate construct system. One component may be an alginate conjugate (or alginate alone) which can include alginate and a protein constituent. The second component may be a salt. Examples of alginate conjugates can include, but are not limited to, alginate-collagen, alginate-laminin, alginate-elastin, alginate-collagen-laminin and alginate-hyaluronic acid in which the collagen, laminin, elastin, collagen-laminin or hyaluronic acid is covalently bonded (or not bonded) to alginate. Examples of salts which can be used to gel the alginate constructs include, but are not limited to, calcium chloride (CaCl2), barium chloride (BaCl2) or strontium chloride (SrCl2).

In one embodiment, the alginate construct is alginate-gelatin. The molecular weight of the gelatin may be in the approximate range of 5 kDa to 100 kDa. The relatively low molecular weight of gelatin offers processing advantages in that it is more soluble and has lower viscosity than hydrogels of higher molecular weight. Another advantage of gelatin is that it contains from 1 to 4 RGD (arginine-glycine-aspartic acid peptide sequence) sites per molecule. RGD is a common cell adhesion ligand and would increase the retention of cells within the infarct zone where the bioscaffolding is formed. The cells retained by the RGD sites may be cells co-injected with the bioscaffolding components or dispersed throughout a component of the system.

The gelatin may be a porcine gelatin or a recombinant human gelatin. The porcine gelatin is a hydrolyzed type 1 collagen extracted from porcine skin. In one embodiment, the molecular weight of the porcine gelatin is approximately 20 kDa. The human gelatin is produced by bacteria using human genetic material. The human recombinant gelatin is equivalent to the porcine gelatin but may reduce the likelihood of an immune response when injected into an infarct region of a human subject.

Alginate is a linear polysaccharide derived from seaweed and contains mannuronic (M) and gluronic acid (G), presented in both alternating blocks and alternating individual residues. It is possible to use some of the carboxyl groups of the alginate as sites to graft useful cell adhesion ligands, such as collagen, laminin, elastin and other peptide fragments of the ECM matrix, forming an alginate conjugate, because alginate does not have RGD groups for cell retention.

An alginate-gelatin conjugate is valuable because it combines characteristics of alginate with characteristics of gelatin, which include, but are not limited to, RGD sites and immunocompatibility. Characteristics of alginate include rapid, almost instantaneous gelation, and an inflammation stimulating effect. The alginate-gelatin conjugate can be formed of approximately 1% to 30% and more particularly approximately 10% to 20% gelatin (either porcine or human recombinant) and approximately 80% to 90% alginate. The relatively lower proportion of alginate-gelatin conjugate is used to retain gelation capacity once combined with pure alginate because the alginate carboxyl groups of alginate that cause the gelation may be bound up in the alginate-gelatin conjugate.

Two-component gelation systems exhibit different characteristics relative to one another including, but not limited to, pore size, storage modulus and gelation time. The gelation system behaves as a sieving media and therefore includes small pores. “Pore size” refers to small, vacuous openings within the gel. “Storage modulus” refers to the strength or the stiffness of the material upon gelation. Storage modulus can be measured by a rheometric instrument. “Gelation time” refers to the kinetics of gelation. Alginate constructs can gel within about 1 second, while fibrin glues can gel between about 5 seconds and about 60 seconds. Self-assembled peptides typically undergo gelation between several minutes to several hours.

In embodiments in which cells are co-injected with the two-component gelation system, or mixed with one component before combining the two components, the gelation system can exhibit different characteristics relative to one another relating to the cells. Such characteristics can include, but are not limited to, morphology of the cells, cell survivability, encapsulation efficiency and/or cell concentration. “Morphology” refers to the physical structure of the cells. In the case of hMSC, the natural morphology is a flattened spindle-shaped morphology. “Cell survivability” is the amount of time that the cells remain viable within the gel post-injection. “Encapsulation efficiency” refers to the fraction of the initial number of cells in suspension that are entrapped within the gel. “Cell concentration” is the encapsulation efficiency divided by the volume of gel formed.

A characteristic which affects the encapsulation efficiency is the difference in viscosity (η) of the two components. If the difference in viscosity between the two components of the gelation system is large, then the encapsulation efficiency is high only when cells are in the high viscosity component. However, if the viscosity of each individual component is lowered without compromising the gelation kinetics, the encapsulation efficiency increases dramatically. For a catheter-based delivery system, low viscosity components are very helpful for successful delivery. A successful application of the two components (which are in solution before delivery) can be dependent upon low viscosity of the individual components.

Modified Gelation Systems

In some embodiments, a bioscaffolding can be formed from a mixture of gel components of different gelation systems. For example, a bioscaffolding can be formed by mixing at least two different components (which do not gel upon mixing under standard cath lab process conditions) of at least two different two-component gelation systems to form a first mixture, and, by mixing at least two different components (other than the components that make up the first mixture and which do not gel upon mixing under standard cath lab process conditions) of the at least two different two-component gelation systems to form a second mixture. “Gel” refers to the phase change from a liquid to a solid upon the combination of two different components or two different mixtures. A treatment agent, such as a cell type or a growth factor, can be added to either the first mixture or the second mixture. The first mixture can then be co-injected with the second mixture to form a bioscaffolding in an infarct region for treatment thereof. In some embodiments, a bioscaffolding can be formed by mixing at least two different gelation components (which do not gel upon mixing under standard cath lab process conditions) to form a first mixture. A treatment agent, such as a cell type or a growth factor, can be added to the first mixture. The first mixture can then be co-injected with a gelation component to form a bioscaffolding on an infarct region for treatment thereof. In some embodiments, the treatment agent can be co-injected with the first mixture or the gelation component without first interdispersing the treatment agent within the first mixture or the gelation component.

In some embodiments, an alginate construct system can include an alginate-gelatin solution as a first component and a calcium chloride solution as a second component. In some embodiments, human mesenchymal stems cells (hMSC) are suspended in one component of the gelation system. hMSC are thought to be capable of both self renewal and differentiation into bone, cartilage, muscle, tendon and fat. hMSC also give rise to a variety of mature cell types via a step-wise maturation process called mesengenesis. The natural morphology of hMSC is elongated and spindle shaped. The gelatin provides RGD sites for cellular adhesion i.e. adhesion of hMSC. Alginate construct systems exhibit rapid gelling kinetics. When combined, alginate-gelatin and calcium chloride gel to form a bioscaffolding in less than 1 second. The resulting gel has a storage modulus of approximately 1 kiloPascal. In application, cell survivability has been observed up to at least 12 days. Encapsulation efficiency is approximately 99%. However, the small pore size of alginate construct systems, which is from about 2 nm to about 500 nm, can lead to low cell spreadability as observed by the round morphology of the hMSC cells over time. “Cell spreading” refers to the naturally occurring morphology of cells. Advantages of alginate construct systems include, but are not limited to, enhanced immune response (a controlled foreign body response) to effect positive remodeling of the injured myocardium, and immunoprotectivity, by shielding via its small pore size, the encapsulated cells from this enhanced immune response (protected from host immune response), instantaneous gelation kinetics, substantial or complete non-adherence to a needle when injected, and long term cell viability. Furthermore, alginate construct systems degrade slowly (at least 8 weeks in vivo).

Fibrin glue can include fibrinogen (modified or not modified with protein constituents) as a first component and thrombin as a second component. In some embodiments, human mesenchymal stems cells (hMSC) are suspended in one component of the gelation system. Fibrin glue systems exhibit fast gelling kinetics, but not as rapid as alginate construct systems. When combined, fibrinogen and thrombin gel to form a bioscaffolding in about 5 seconds to about 10 seconds. The resulting gel has a storage modulus of approximately 3 kiloPascals which is higher than that of alginate construct systems. A higher storage modulus may improve mechanical reinforcement at the infarct region. In application, cell survivability has been observed up to at least 12 days. The pore size of fibrin glue systems is from about 1.5 microns to about 2.5 microns and can lead to high cell spreadability of hMSC cells. That is, hMSC cells have been observed to have an elongate and stellate morphology which is more natural to their endogenous state when compared to the morphology observed in alginate construct systems alone. Advantages of fibrin glue include, but are not limited to, material strength, promotion of angiogenesis, good cytocompatibility (compatible with cell growth), good cell morphology and high cell proliferation in fibrinogen.

The following is a table comparing experimental data and characteristics of alginate construct and fibrin glue systems:

TABLE 1
1% Alginate-collagen in 100% Tiseel ™
Characteristic 2% CaCl2 (no dilution)
Pore size 200-500 nm 1.5-2.5 μm
Storage modulus 1 kPa 3 kPa
Gelation kinetics 1 second 5-10 seconds
Encapsulation efficiency 99.26 ± 0.61 93.71 ± 0.61
Morphology spherical elongated,
spindle-shaped
within 24 hours
Cell survivability 12 days to 6 months at least 12 days
Degradation time in vivo 8 weeks 2 weeks

In some embodiments, a bioscaffolding is formed from mixing components of at least two gelation systems. For example, a first component of a first two-component gel and a first component of a second two-component gel can be combined to form a first mixture. A second component of a first two-component gel and a second component of the second two-component gel can be combined to form a second mixture. Cells can be suspended within either the first mixture or the second mixture. When the two mixtures are combined, a bioscaffolding including at least some advantageous characteristics of both gelation systems can be realized. In some embodiments, a bioscaffolding can be formed by mixing at least two different gelation components to form a first mixture. When the first mixture is combined with a gelation salt, a bioscaffolding including at least some advantageous characteristics of the individual components can be realized. It should be appreciated that a number of different combinations of gelation components can be mixed together in different ratios to accentuate various advantageous characteristics of the individual gelation systems. Furthermore, the concentration of the individual components, either singly or combined, can influence certain characteristics of the bioscaffolding, such as viscosity and encapsulation efficiency.

Alginate Construct Systems and Fibrin Glues

Experiment 1

hMSC were suspended in a 1% alginate-collagen solution at 1.43×107 cells/mL. 200 microliters of the alginate-collagen solution was combined with 200 microliters of a thrombin solution containing 40 mM CaCl2. Encapsulation efficiency of the resulting gel was measured at 99.16±0.007%. Encapsulation efficiency was measured as the difference between the initial number of cells in the suspension and the number of cells remaining in the supernatant divided by the initial number of cells and expressed as a percentage. On day 3, the cells exhibited a round morphology. On day 7 and 12, the cells continued to exhibit a round morphology. Cells were analyzed by a molecular probe LIVE/DEAD® Assay available from Invitrogen, Inc. The viscosity of the suspension component was measured at approximately 33 centipoise (cp).

Experiment 2

hMSC were suspended in a thrombin solution containing 40 mM of CaCl2 at a concentration of 2.12×107 cells/mL. A 1% solution of alginate-collagen was combined with the thrombin solution in a 1:1 ratio (200 μL:200 μL). Encapsulation efficiency of the resulting gel was measured at 26.47±5.5%. The viscosity of the suspension component was measured at approximately 7 cp.

Experiment 3

A fibrin glue kit Tisseel™ including modified fibrinogen and thrombin was obtained from Baxter. hMSC were suspended in a thrombin solution at a concentration of 2.96×107 cells/mL. 200 microliters of the thrombin solution containing cells was combined with the fibrinogen solution in a 1:1 ratio. Encapsulation efficiency of the resulting gel was measured at 93.71±3.48%. On day 3, the cells exhibited an elongated, spindle shaped morphology. On day 7 & 12 the cells continued to exhibit similar elongated and spindle shaped morphology and there was visible but no quantitative evidence of cell proliferation. Cells were analyzed by a molecular probe LIVE/DEAD® Assay available from Invitrogen, Inc. The viscosity of the suspension component was measured at approximately 7 cp.

Experiment 4

A fibrin glue kit Tisseel™ including modified fibrinogen and thrombin was obtained from Baxter. Fibrinogen was reconstituted as directed in accompanying instructions and then further diluted with water to half of its original concentration. hMSC were suspended in the reconstituted, full strength thrombin solution at a concentration of 2.96×107 cells/mL. 200 microliters of the thrombin solution containing hMSC was combined with the diluted fibrinogen solution in a 1:1 ratio. Encapsulation efficiency of the resulting gel was measured at 98.07±1.6%. The viscosity of the suspension component was measured at approximately 7 cp.

Experiment 5

A fibrin glue kit Tisseel™ including modified fibrinogen and thrombin was obtained from Baxter. Fibrinogen was reconstituted as directed in accompanying instructions and then further diluted with water to half of its original concentration. hMSC were suspended in the diluted fibrinogen solution at a concentration of 2.96×107 cells/mL. 200 microliters of the diluted fibrinogen solution containing hMSC was combined with the thrombin solution in a 1:1 ratio. Encapsulation efficiency of the resulting gel was measured at 97.12±1.73%. The viscosity of the suspension component was measured at approximately 5 cp.

Experiment 6

A fibrin glue kit Tisseel™ including modified fibrinogen and thrombin was obtained from Baxter. Fibrinogen was reconstituted as directed and then further diluted with water to half of its original concentration. The diluted fibrinogen solution was mixed with a 0.5% alginate-collagen solution in a 1:1 ratio to form a first mixture. hMSC were suspended in a thrombin solution (which contains 40 mM CaCl2) at a concentration of 2.96×107 cells/mL. 200 microliters of the first mixture was combined with the thrombin in a 1:1 ratio. Encapsulation efficiency of the resulting gel was measured at 91.39±6.78%. The viscosity of the suspension component was measured at approximately 7 cp.

Experiment 7

A fibrin glue kit Tisseel™ including modified fibrinogen and thrombin was obtained from Baxter. Fibrinogen was reconstituted as directed and then further diluted with water to half of its original concentration. The diluted fibrinogen solution was mixed with a 0.5% alginate-collagen solution in a 1:1 ratio to form a first mixture. hMSC were suspended in the first mixture at a concentration of 5.51×107 cells/mL. The first mixture was combined with the second mixture comprising thrombin and a 2% CaCl2 solution in a 1:1 ratio. Encapsulation efficiency of the resulting gel was measured at 99.42+0.12%. The viscosity of the suspension component was measured at approximately 6 cp.

Table 2 summarizes the results of a table of experimental data showing the various characteristics of combinations of alginate construct and fibrin glue systems:

TABLE 2
Component
First Second in which Viscosity of
component/first component/second Encapsulation cells component
mixture mixture Efficiency suspended (centipoise)
1 1% alginate- Thrombin  99.16 ± 0.007 alginate- 33.28 ± 2.3 
collagen containing 40 mM collagen
CaCl2
2 1% alginate- Thrombin 26.47 ± 5.5  thrombin 7.32 ± 2.6
collagen containing 40 mM
CaCl2
3 Undiluted Thrombin 93.71 ± 3.48 thrombin 7.32 ± 2.6
fibrinogen containing 40 mM
CaCl2
4 Diluted Thrombin 98.07 ± 1.6  thrombin 7.32 ± 2.6
fibrinogen containing 40 mM
CaCl2
5 Diluted Thrombin 97.12 ± 1.73 fibrinogen  5.4 ± 2.8
fibrinogen containing 40 mM
CaCl2
6 Diluted Thrombin 91.39 ± 6.78 thrombin 7.32 ± 2.6
fibrinogen + containing 40 mM
0.5% alginate- CaCl2
collagen
7 Diluted Thrombin 99.42 ± 0.12 5%  6.5 ± 3.2
fibrinogen + containing 40 mM fibrinogen +
0.5% alginate- CaCl2 0.5%
collagen alginate-
collagen

In one embodiment, a bioscaffolding can be formed by mixing components of more than one gelation system. For example, alginate-gelatin (of an alginate construct system) can be mixed with fibrinogen (of a fibrin glue system) in a 1:1 ratio to form a first mixture. Thrombin (of a fibrin glue system) already contains 40 mM calcium chloride (which is required to gel an alginate construct system) to form a second mixture. Experiments 6 and 7 may represent such a combination. hMSC can be added to an individual component or to the first or second mixture.

Experiment 3 illustrates that, in a standard two-component gelation system which includes a first component (alginate) or a second component (thrombin) only, if the hMSC are suspended in a low viscosity component, i.e., thrombin (η=7.32±2.6), encapsulation efficiency is very low (E=26.47±5.5%)(i.e., alginate-collagen). However, note that in these experiments, the viscosity of the other component is high and there is a wide mismatch between the two viscosities e.g., the viscosity of 1% alginate-collagen is 33.2%+2.3%. Further, alginate has faster gelation kinetics than the other systems. In experiments 6 and 7 however, even though the hMSC were suspended in a low viscosity component or mixture, the resulting gel showed high encapsulation efficiency. It is theorized that this result is due to the viscosity of the other component being low as well and that there was not much of a mismatch in viscosities between the two components, thus allowing good mixing. Further, the mixed systems gel slower allowing more cell diffusion from the lower viscosity component to the gel. Thus, it has been shown that even if the individual components in which the hMSC is suspended have low viscosities (before gelation), the resulting gel can still exhibit a high encapsulation efficiency (after gelation). This is highly advantageous, as lower viscosity may result in a more successful delivery with a catheter-based delivery system because, for example, such a system requires less force for injection into the infarct region and higher flow rates can be achieved. Additionally, lower viscosity solutions may result in less residual within the lumens of a delivery system. The combination also may provide a high encapsulation efficiency while allowing hMSC to achieve a natural state of morphology, i.e., elongated and spindle shaped. Thus, such a bioscaffolding may include at least some advantageous characteristics of the individual components comprising the two-component gelation systems.

In another embodiment, a bioscaffolding can be formed by mixing alginate-gelatin with sodium-hyaluronate and gelled with calcium chloride. The hyaluronate will be immobilized by chain entanglement and provide attachment ligands for stem cells bearing the CD44 receptor, e.g., human mesenchymal stem cells. Appropriate formulations can have 50% to 99% of a 0.5% to 1.0% solution of alginate-gelatin combined with 1% to 50% of a 0.05% to 1% solution of sodium hyaluronate (Genzyme Biosurgery, Mass.). The mixture can be gelled by the addition of an equal volume of a 0.5% to 1.5% of calcium chloride dihydrate.

In another embodiment, a bioscaffolding can be formed by mixing reconstituted lyophilized peptide (with buffer) with alginate and gelled with calcium chloride. Examples of peptides include self-assembled peptides, such as RAD 16-II (SEQ ID NO: 1), MAX-1 (SEQ ID NO: 2) and EAK16-II (SEQ ID NO: 3). These peptides gel when exposed to conditions of physiological or greater osmolarity, at neutral pH. However, gelation kinetics are relatively slow, ranging from minutes to hours. Introduction of these peptides as a sole scaffold would result in an intercalated structure, at best, due to the slow gelation. Addition of alginate can provide rapid gelation kinetics, thus preventing dissipation of the peptide into tissue.

In some embodiments, a self-assembled peptide (SAP) component can be mixed with a growth factor to form a first mixture. Examples of self-assembled peptides include RAD 16-II (SEQ ID NO: 1), MAX-1 (SEQ ID NO: 2) and EAK16-II (SEQ ID NO: 3). Examples of growth factors in include, but are not limited to, isoforms of vasoendothelial growth factor (VEGF), fibroblast growth factor (FGF, e.g. beta-FGF), Del 1, hypoxia inducing factor (HIF 1-alpha), monocyte chemoattractant protein (MCP-1), nicotine, platelet derived growth factor (PDGF), insulin-like growth factor 1 (IGF-1), transforming growth factor (TGF alpha), hepatocyte growth factor (HGF), estrogens, follistatin, proliferin, prostaglandin E1 and E2, tumor necrosis factor (TNF-alpha), interleukin 8 (Il-8), hematopoietic growth factors, erythropoietin, granulocyte-colony stimulating factors (G-CSF) and platelet-derived endothelial growth factor (PD-ECGF). The first mixture can be combined with a component of a two-component gelation system, such as an alginate construct system, a fibrin glue or a polymer system, or any combination thereof. In some embodiments, human mesenchymal stem cells can be added to the system.

In one embodiment, the self-assembled peptide is RAD 16-II (SEQ ID NO: 1) and the growth factor is PDGF or a derivative thereof and combines to form a first mixture. It has been shown that PDGF mediates cardiac microvascular endothelial cell communication with cardiomyocytes and it is anticipated that an application of PDGF can restore damaged endothelial PDGF-regulated angiogenesis and enhance post-ischemic neovascularization in an infarct region. When combined with RAD 16-11 (SEQ ID NO: 1), PDGF binds to RAD 16-II (SEQ ID NO: 1) through weak molecular interactions. In some applications, PDGF remains viable for approximately fourteen days when combined with RAD 16-11 (SEQ ID NO: 1) when applied to an infarct region. However, it is anticipated that the slow kinetics of the self-assembled peptide, i.e., minutes to hours, will cause significant leakage, backflow and dissipation before the peptide can form a nanofiber bioscaffolding at the infarct region.

In some embodiments, the first mixture (comprising RAD 16-II (SEQ ID NO: 1) and PDGF) can be combined with any one component of fibrin glue or an alginate construct. The rapid kinetics of both fibrin glue and alginate constructs can counteract the slow kinetics of the SAP-PDGF construct. It is therefore anticipated that leakage and dissipation at the infarct region can be reduced by combining the SAP-PDGF construct with any one component of fibrin glue or an alginate construct.

Delivery Systems

Devices which can be used to deliver modified or combined components of the gelation systems include, but are not limited to, dual-needle left-ventricle injection devices, dual-needle transvascular wall injection devices and dual syringes. Methods of access to use the minimally invasive (i.e., percutaneous or endoscopic) injection devices include access via the femoral artery or the sub-xiphoid. “Xiphoid” or “xiphoid process” is a pointed cartilage attached to the lower end of the breastbone or sternum, the smallest and lowest division of the sternum. Both methods are known by those skilled in the art.

FIG. 2 illustrates an embodiment of a dual syringe device which can be used to deliver the compositions of the present invention. Dual syringe 400 can include first barrel 410 and second barrel 420 adjacent to one another and connected at a proximal end 455, distal end 460 and middle region 465 by plates 440, 445 and 450, respectively. In some embodiments, barrels 410 and 420 can be connected by less than three plates. Each barrel 410 and 420 includes plunger 415 and plunger 425, respectively. Barrels 410 and 420 can terminate at a distal end into needles 430 and 435, respectively, for extruding a substance. In some embodiments, barrels 410 and 420 can terminate into cannula protrusions for extruding a substance. Barrels 410 and 420 should be in close enough proximity to each other such that the substances in each respective barrel are capable of mixing with one another to form a bioscaffolding in the treatment area, e.g., a post-infarct myocardial region. Dual syringe 400 can be constructed of any metal or plastic which is minimally reactive or completely unreactive with the formulations described in the present invention. In some embodiments, dual syringe 400 includes a pre-mixing chamber attached to distal end 465.

In some applications, first barrel 410 can include a first mixture of a modified two-component gelation system and second barrel 420 can include a second mixture of a modified two-component gelation system according to any of the embodiments described previously. A therapeutic amount of the resulting gel is between about 25 μL to about 200 μL, preferably about 50 μL. Dual syringe 400 can be used during, for example, an open chest surgical procedure.

FIGS. 3A-3B illustrate an embodiment of a dual-needle injection device which can be used to deliver the compositions of the present invention. Delivery assembly 500 includes lumen 510 which may house delivery lumens, guidewire lumens and/or other lumens. Lumen 510, in this example, extends between distal portion 505 and proximal end 515 of delivery assembly 500.

In one embodiment, delivery assembly 500 includes first needle 520 movably disposed within delivery lumen 530. Delivery lumen 530 is, for example, a polymer tubing of a suitable material (e.g., polyamides, polyolefins, polyurethanes, etc.). First needle 520 is, for example, a stainless steel hypotube that extends a length of the delivery assembly. First needle 520 includes a lumen with an inside diameter of, for example, 0.08 inches (0.20 centimeters). In one example for a retractable needle catheter, first needle 520 has a needle length on the order of about 40 inches (about 1.6 meters) from distal portion 505 to proximal portion 515. Lumen 510 also includes auxiliary lumen 540 extending, in this example, co-linearly along the length of the catheter (from a distal portion 505 to proximal portion 515). Auxiliary lumen 540 is, for example, a polymer tubing of a suitable material (e.g., polyamides, polyolefins, polyurethanes, etc.). At distal portion 505, auxiliary lumen 540 is terminated at a delivery end of second needle 550 and co-linearly aligned with a delivery end of needle 520. Auxiliary lumen 540 may be terminated to a delivery end of second needle 550 with a radiation-curable adhesive, such as an ultraviolet curable adhesive. Second needle 550 is, for example, a stainless steel hypotube that is joined co-linearly to the end of main needle 520 by, for example, solder (illustrated as joint 555). Second needle 550 has a length on the order of about 0.08 inches (0.20 centimeters). FIG. 3B shows a cross-sectional front view through line A-A′ of delivery assembly 500. FIG. 3B shows main needle 520 and second needle 550 in a co-linear alignment.

Referring to FIG. 3A, at proximal portion 515, auxiliary lumen 540 is terminated to auxiliary side arm 560. Auxiliary side arm 560 includes a portion extending co-linearly with main needle 520. Auxiliary side arm 560 is, for example, a stainless steel hypotube material that may be soldered to main needle 520 (illustrated as joint 565). Auxiliary side arm 560 has a co-linear length on the order of about, in one example, 1.2 inches (3 centimeters).

The proximal end of main needle 520 includes adaptor 570 for accommodating a substance delivery device. Adaptor 570 is, for example, a molded female luer housing. Similarly, a proximal end of auxiliary side arm 560 includes adaptor 580 to accommodate a substance delivery device (e.g., a female luer housing).

The design configuration described above with respect to FIGS. 3A-3B is suitable for introducing modified two-component gel compositions of the present invention. For example, a gel may be formed by a combination (mixing, contact, etc.) of a first mixture of a modified two-component gelation system and a second mixture of a modified two-component gelation system. Representatively, a first mixture may be introduced by a one cubic centimeters syringe at adaptor 570 through main needle 520. At the same time or shortly before or after, a second mixture may be introduced with a one cubic centimeter syringe at adaptor 580. When the first and second components combine at the exit of delivery assembly 500 (at an infarct region), the materials combine (mix, contact) to form a bioerodible gel.

FIGS. 4A-4C illustrate an alternative embodiment of a dual-needle injection device which can be used to deliver two-component gel compositions of the present invention. In general, the catheter assembly 600 provides a system for delivering substances, such as modified two-component gel compositions, to or through a desired area of a blood vessel (a physiological lumen) or tissue in order to treat a myocardial infarct region. The catheter assembly 600 is similar to the catheter assembly described in commonly-owned, U.S. Pat. No. 6,554,801, titled “Directional Needle Injection Drug Delivery Device”, which is incorporated herein by reference.

In one embodiment, catheter assembly 600 is defined by elongated catheter body 650 having proximal portion 620 and distal portion 610. Guidewire cannula 670 is formed within catheter body (from proximal portion 610 to distal portion 620) for allowing catheter assembly 600 to be fed and maneuvered over guidewire 680. Balloon 630 is incorporated at distal portion 610 of catheter assembly 600 and is in fluid communication with inflation cannula 660 of catheter assembly 600.

Balloon 630 can be formed from balloon wall or membrane 635 which is selectively inflatable to dilate from a collapsed configuration to a desired and controlled expanded configuration. Balloon 630 can be selectively dilated (inflated) by supplying a fluid into inflation cannula 660 at a predetermined rate of pressure through inflation port 665 (located at proximal end 620). Balloon wall 635 is selectively deflatable, after inflation, to return to the collapsed configuration or a deflated profile. Balloon 630 may be dilated (inflated) by the introduction of a liquid into inflation cannula 660. Liquids containing treatment and/or diagnostic agents may also be used to inflate balloon 630. In one embodiment, balloon 630 may be made of a material that is permeable to such treatment and/or diagnostic liquids. To inflate balloon 630, the fluid can be supplied into inflation cannula 660 at a predetermined pressure, for example, between about one and 20 atmospheres. The specific pressure depends on various factors, such as the thickness of balloon wall 635, the material from which balloon wall 635 is made, the type of substance employed and the flow-rate that is desired.

Catheter assembly 600 also includes at least two substance delivery assemblies (not shown; see FIGS. 4B-4C) for injecting a substance into a myocardial infarct region. In one embodiment, substance delivery assembly 605 a includes needle 615 a movably disposed within hollow delivery lumen 625 a. Delivery assembly 605 b includes needle 615 b movably disposed within hollow delivery lumen 625 b (not shown; see FIGS. 4B-4C). Delivery lumen 625 a and delivery lumen 625 b each extend between distal portion 610 and proximal portion 620. Delivery lumen 625 a and delivery lumen 625 b can be made from any suitable material, such as polymers and copolymers of polyamides, polyolefins, polyurethanes and the like. Access to the proximal end of delivery lumen 625 a or delivery lumen 625 b for insertion of needle 615 a or 615 b, respectively is provided through hub 635 (located at proximal end 620). Delivery lumens 625 a and 625 b may be used to deliver first and second mixtures of a modified two-component gel composition to a post-myocardial infarct region.

FIG. 4B shows a cross-section of catheter assembly 600 through line A-A′ of FIG. 4A (at distal portion 610). FIG. 4C shows a cross-section of catheter assembly 600 through line B—B′ of FIG. 4A. In some embodiments, delivery assemblies 605 a and 605 b are adjacent to each other. The proximity of delivery assemblies 605 a and 605 b allows each mixture of the modified two-component gelation system to rapidly gel when delivered to a treatment site, such as a post-myocardial infarct region.

From the foregoing detailed description, it will be evident that there are a number of changes, adaptations and modifications of the present invention which come within the province of those skilled in the part. The scope of the invention includes any combination of the elements from the different species and embodiments disclosed herein, as well as subassemblies, assemblies and methods thereof. However, it is intended that all such variations not departing from the spirit of the invention be considered as within the scope thereof.

Claims (16)

What is claimed is:
1. A composition comprising:
a first mixture comprising a first component of a first two-component gel system and a first component of a second two-component gel system,
wherein the first component of the first two component gel system is one of an alginate-collagen, an alginate-laminin, an alginate-elastin, an alginate-collagen-laminin, an alginate-hyaluronic acid, an alginate and a self-assembled peptide, and wherein the first component of the second two-component gel system is one of fibrinogen, a fibrinogen conjugate, and sodium hyaluronate, and
a second mixture comprising a second component of the first two-component gel system and a second component of the second two-component gel system, wherein the second component of the first two-component gel system is one of sodium chloride, calcium chloride, barium chloride, strontium chloride and sucrose, and wherein the second component of the second two-component gel system is different than the second component of the first two-component gel system, and is one of thrombin and sucrose, and
wherein upon separately injecting the first mixture and the second mixture into a post-myocardial infarction area of a heart, the first component and the second component of the first two-component gel system mix to form a first gel and the first component and the second component of the second two-component gel system mix to form a second gel, and
wherein the first gel has a different characteristic than the second gel.
2. The composition of claim 1, further comprising at least one cell type suspended in one of the first mixture or the second mixture.
3. The composition of claim 2, wherein the cell type is selected from the group consisting of localized cardiac progenitor cells, mesenchymal stem cells, bone marrow derived mononuclear cells, adipose stem cells, embryonic stem cells, umbilical cord blood derived stem cells, smooth muscle cells and skeletal myoblasts.
4. The composition of claim 2, further comprising at least one growth factor in one of the first mixture or the second mixture.
5. The composition of claim 4, wherein the growth factor is selected from the group consisting of vasoendothelial growth factor, fibroblast growth factor, Del 1, hypoxia inducing factor, monocyte chemoattractant protein, nicotine, platelet derived growth factor, insulin-like growth factor 1, transforming growth factor, hepatocyte growth factor, estrogens, follistatin, proliferin, prostaglandin E1 and E2, tumor necrosis factor, interleukin 8, hematopoietic growth factors, erythropoietin, granulocyte-colony stimulating factors and platelet-derived endothelial growth factor.
6. The composition of claim 1, wherein the first component of the first two-component gel system and the second component of the second two-component gel system is mixed in a one-to-one ratio.
7. The composition of claim 1, wherein (a) the first component of the first two-component gel system is an alginate-collagen solution and the first component of the second two-component gel system is a fibrinogen solution and (b) the second component of the first two-component gel system is a 2.0% mass per volume calcium chloride solution and the second component of the second two-component gel system is a thrombin solution.
8. The composition of claim 7, further comprising human mesenchymal stem cells suspended in one of the first mixture or the second mixture.
9. The composition of claim 7, wherein the concentration of alginate-collagen is in a range from between 0.5% mass per volume to 1.0% mass per volume.
10. The composition of claim 7, wherein the concentration of fibrinogen is in a range from between 5.0% mass per volume to 7.0% mass per volume.
11. The composition of claim 7, wherein the concentration of thrombin is in a range from between 9.5% mass per volume to 10.0% mass per volume.
12. The composition of claim 7, wherein the alginate-collagen and fibrinogen are mixed in a one-to-one ratio.
13. The composition of the claim 7, wherein the thrombin and calcium chloride are mixed in a one-to-one ratio.
14. The composition of claim 1, wherein (a) the first component of the first two-component gel is a self-assembled peptide and the first component of the second two-component gel is fibrinogen and (b) the second component of the first two-component gel is 2.0% mass per volume calcium chloride and the second component of the second two-component gel is thrombin.
15. The composition of claim 14, further comprising platelet derived growth factor combined with the self-assembled peptide.
16. The composition of claim 14, wherein the self-assembled peptide is selected from the group consisting of RAD 16-II (SEQ ID NO: 1), MAX-1 (SEQ ID NO: 2) and EAK16-II (SEQ ID NO: 3).
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